Mycobacterium tuberculosis (Mtb) is a pathogen of tremendous global importance that can persist for years in both latent and active infections, complicating eradication efforts and necessitating long treatment regimens. The mechanisms employed by Mtb to adapt to and withstand fluctuating in vivo conditions and host immune attack must be better understood in order to facilitate the rational design of therapeutics. DNA methylation plays important regulatory roles in several prokaryotic pathogens but has not been comprehensively addressed in mycobacteria. The proposed work will investigate the role of a DNA methyltransferase that has been implicated in virulence and resistance to hypoxia in two separate genetic screens. The objective of this work is to determine the functional consequences of methylation in Mtb, with the long-term goal of furthering understanding of the mechanisms of pathogenesis of this pathogen so that appropriate targets for intervention can be identified. Specific aims: (1) Comprehensively identify sites of adenine methylation by Mrh (Methyltransferase Required in Hypoxia) in the Mtb genome; (2) Test the functional significance of Mrh-mediated methylation in Mtb by assessing the virulence and hypoxia resistance of an mrh deletion strain; and (3) Elucidate the molecular consequences of Mrh-mediated methylation in Mtb. Study design: The methylation consensus sequence determined in preliminary studies will be used to map Mrh sites across the genome and predict loci that may be affected by methylation. Mrh deletion strains will be used to test the hypotheses, suggested by transposon screening data, that (1) Mrh-mediated methylation is required for Mtb to survive in hypoxia, an in vitro condition that may mimic the intracellular and intragranuloma environments, and (2) that Mrh-mediated methylation is important for growth in macrophages. Deletion strains will be further employed to test the hypothesis that Mrh-mediated methylation is important for virulence of Mtb in a mouse model of infection. Whole genome expression profiling will be performed under a series of relevant conditions to determine if Mrh-mediated methylation plays a role in the regulation of gene expression. Analyses at the single-cell level and of individual loci will follow. Alternative hypotheses for the function of Mrh such as control of cell cycle timing, protection of DNA from damaging agents, and roles in DNA repair will also be explored. These studies will be extended to investigate the functional significance of a point mutation in mrh that is present in an important clinical Mtb lineage. The proposed work is expected to provide insight into the role and significance of methylation in Mtb, a topic that has never before been investigated but is suggested by genetic screens to be important for this pathogen.